DNA demethylation in vitro: involvement of RNA

An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 myoblasts specifically demethylate an alpha-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.